SUMMARY- from the parent grant 1R01GM136905-01 The human LonP1 protease is a master regulator of mitochondrial proteostasis, which is essential for regulating mitochondrial energy metabolism and mitigating cell stress. We recently identified a novel pathogenic variant in the LONP1 gene encoding the protease, in two siblings with profound neurologic impairment, cerebral and cerebellar atrophy- proline at position 761 was replaced by leucine (Lon- P761L). Primary skin fibroblasts from these siblings, showed substantially reduced activity of pyruvate dehydrogenase (PDH). We showed that deficiency of PDH was caused by the failure of mutant Lon- P761L to degrade a subunit of PDH- phosphorylated E1a, which accumulates and inhibits PDH activity. PDH is the central gatekeeper linking glycolysis to the tricarboxylic acid (TCA) cycle and it is also a key node for regulating glucose and fatty acid catabolism. Our long term goal is to elucidate the crucial role of LonP1 in the regulation of energy metabolism, and why homozygous Lon-P761L expression causes severe neurologic dysfunction and neurodegeneration. Glucose is the brain?s principal source of energy. Neurons generate ATP almost exclusively by glucose oxidization, thus fully functional PDH activity is crucial. Astrocytes by contrast, have broader metabolic capacity and supply neurons with lactate, glutamine and ketone bodies, which are used to form acetyl CoA and TCA cycle intermediates required for glucose oxidation. We hypothesize that wild type LonP1 regulates the architecture and activities of the PDH complex, and modulates upstream and downstream effectors, to calibrate mitochondrial metabolism and energetics. In this project, we will employ patient- and parent- derived fibroblasts, and also fibroblasts that have been reprogrammed to generate induced pluripotent stem cells (iPSCs). These iPSCs will be differentiated into neurons and astrocytes. Using the patient- and parent- derived fibroblasts, Aim 1 will test the hypothesis that LonP1-mediated degradation regulates the architecture and activity of the PDH complex. Aim 2 will identify the up- and down- stream modulators of the LonP1- PDH axis, which are altered in cells expressing wild type LonP1 versus Lon-P761L. In Aim 3, we will investigate the regulation of PDH by LonP1 in iPSCs differentiated into neurons and astrocytes. Our investigation will establish new molecular mechanisms for the Lon-dependent regulation of PDH. The knowledge gained will also help to identify potential therapeutic protein targets (e.g. PDK, PDP, LonP1), pharmacologic and dietary interventions for increasing PDH activity and/or for treating PDH deficiency associated with LonP1 dysfunction. These outcomes will have a broader impact for understanding how PDH activity and mitochondrial metabolism can be calibrated in rare and also more common disorders such as heart disease, cancer and neurodegeneration.